Activation of muscarinic receptors in PC12 cells. Stimulation of Ca2+ influx and redistribution

T. Pozzan, F. Di Virgilio, L. M. Vicentini, J. Meldolesi

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Abstract

Ca2+ homoeostasis was investigated in pheochromocytoma neurosecretory (PC12) cells both before and after treatment with nerve growth factor, which induces a neuronal-like differentiation accompanied by a large increase in the number of muscarinic receptors. The resting concentration of free cytosolic Ca2+, [Ca2+](i), measured by the quin2 technique, was found to be higher and more variable in differentiated cells. Moreover, the [Ca2+](i) rises induced by the Ca2+ ionophore ionomycin and by depolarizing concentrations of KCl were greater and more transient. Exposure to carbachol induced modest, but long-lasting, [Ca2+](i) rises, which were faster and greater in differentiated than in non-differentiated cells. These effects were due to the activation of the muscarinic receptor, because they were unaffected by nicotinic blockers (hexamethonium and D-tubocurarine) and completely eliminated by low concentrations of the muscarinic antagonists atropine and pirenzepine [IC50 (concn. causing 50% inhibition) = 2 and 60 nM respectively]. The muscarinic-receptor-dependent [Ca2+](i) rises were the result of two concomitant processes: (1) redistribution of Ca2+ from cytoplasmic stores to the cytosol, possibly mediated by generation of inositol 1,4,5-trisphosphate as a consequence of the muscarinic-receptor-coupled hydrolysis of polyphosphoinositides, and (2) increased Ca2+ influx through a pathway of the plasmalemma insensitive to verapamil and thus different from the voltage-dependent Ca2+ channel. The existence of this second process was documented: (a) by the difference of the [Ca2+](i) responses brought about by carbachol in Ca2+-containing and Ca2+-free media; (b) by the occurrence of [Ca2+](i) rise and increased 45Ca accumulation in cells exposed to 1 mM-CaCl2 after having been treated for 2 min with carbachol in Ca2+-free medium; (c) by typical differences in the quin2 signal kinetics observed in parallel samples of PC12 cells loaded with different concentrations of the dye.

Original languageEnglish
Pages (from-to)547-553
Number of pages7
JournalBiochemical Journal
Volume234
Issue number3
Publication statusPublished - 1986

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PC12 Cells
Muscarinic Receptors
Carbachol
Chemical activation
Pirenzepine
Phosphatidylinositol Phosphates
Tubocurarine
Hexamethonium
Ionomycin
Muscarinic Antagonists
Inositol 1,4,5-Trisphosphate
Ionophores
Pheochromocytoma
Nerve Growth Factor
Verapamil
Atropine
Cytosol
Inhibitory Concentration 50
Hydrolysis
Homeostasis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Pozzan, T., Di Virgilio, F., Vicentini, L. M., & Meldolesi, J. (1986). Activation of muscarinic receptors in PC12 cells. Stimulation of Ca2+ influx and redistribution. Biochemical Journal, 234(3), 547-553.

Activation of muscarinic receptors in PC12 cells. Stimulation of Ca2+ influx and redistribution. / Pozzan, T.; Di Virgilio, F.; Vicentini, L. M.; Meldolesi, J.

In: Biochemical Journal, Vol. 234, No. 3, 1986, p. 547-553.

Research output: Contribution to journalArticle

Pozzan, T, Di Virgilio, F, Vicentini, LM & Meldolesi, J 1986, 'Activation of muscarinic receptors in PC12 cells. Stimulation of Ca2+ influx and redistribution', Biochemical Journal, vol. 234, no. 3, pp. 547-553.
Pozzan T, Di Virgilio F, Vicentini LM, Meldolesi J. Activation of muscarinic receptors in PC12 cells. Stimulation of Ca2+ influx and redistribution. Biochemical Journal. 1986;234(3):547-553.
Pozzan, T. ; Di Virgilio, F. ; Vicentini, L. M. ; Meldolesi, J. / Activation of muscarinic receptors in PC12 cells. Stimulation of Ca2+ influx and redistribution. In: Biochemical Journal. 1986 ; Vol. 234, No. 3. pp. 547-553.
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abstract = "Ca2+ homoeostasis was investigated in pheochromocytoma neurosecretory (PC12) cells both before and after treatment with nerve growth factor, which induces a neuronal-like differentiation accompanied by a large increase in the number of muscarinic receptors. The resting concentration of free cytosolic Ca2+, [Ca2+](i), measured by the quin2 technique, was found to be higher and more variable in differentiated cells. Moreover, the [Ca2+](i) rises induced by the Ca2+ ionophore ionomycin and by depolarizing concentrations of KCl were greater and more transient. Exposure to carbachol induced modest, but long-lasting, [Ca2+](i) rises, which were faster and greater in differentiated than in non-differentiated cells. These effects were due to the activation of the muscarinic receptor, because they were unaffected by nicotinic blockers (hexamethonium and D-tubocurarine) and completely eliminated by low concentrations of the muscarinic antagonists atropine and pirenzepine [IC50 (concn. causing 50{\%} inhibition) = 2 and 60 nM respectively]. The muscarinic-receptor-dependent [Ca2+](i) rises were the result of two concomitant processes: (1) redistribution of Ca2+ from cytoplasmic stores to the cytosol, possibly mediated by generation of inositol 1,4,5-trisphosphate as a consequence of the muscarinic-receptor-coupled hydrolysis of polyphosphoinositides, and (2) increased Ca2+ influx through a pathway of the plasmalemma insensitive to verapamil and thus different from the voltage-dependent Ca2+ channel. The existence of this second process was documented: (a) by the difference of the [Ca2+](i) responses brought about by carbachol in Ca2+-containing and Ca2+-free media; (b) by the occurrence of [Ca2+](i) rise and increased 45Ca accumulation in cells exposed to 1 mM-CaCl2 after having been treated for 2 min with carbachol in Ca2+-free medium; (c) by typical differences in the quin2 signal kinetics observed in parallel samples of PC12 cells loaded with different concentrations of the dye.",
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AB - Ca2+ homoeostasis was investigated in pheochromocytoma neurosecretory (PC12) cells both before and after treatment with nerve growth factor, which induces a neuronal-like differentiation accompanied by a large increase in the number of muscarinic receptors. The resting concentration of free cytosolic Ca2+, [Ca2+](i), measured by the quin2 technique, was found to be higher and more variable in differentiated cells. Moreover, the [Ca2+](i) rises induced by the Ca2+ ionophore ionomycin and by depolarizing concentrations of KCl were greater and more transient. Exposure to carbachol induced modest, but long-lasting, [Ca2+](i) rises, which were faster and greater in differentiated than in non-differentiated cells. These effects were due to the activation of the muscarinic receptor, because they were unaffected by nicotinic blockers (hexamethonium and D-tubocurarine) and completely eliminated by low concentrations of the muscarinic antagonists atropine and pirenzepine [IC50 (concn. causing 50% inhibition) = 2 and 60 nM respectively]. The muscarinic-receptor-dependent [Ca2+](i) rises were the result of two concomitant processes: (1) redistribution of Ca2+ from cytoplasmic stores to the cytosol, possibly mediated by generation of inositol 1,4,5-trisphosphate as a consequence of the muscarinic-receptor-coupled hydrolysis of polyphosphoinositides, and (2) increased Ca2+ influx through a pathway of the plasmalemma insensitive to verapamil and thus different from the voltage-dependent Ca2+ channel. The existence of this second process was documented: (a) by the difference of the [Ca2+](i) responses brought about by carbachol in Ca2+-containing and Ca2+-free media; (b) by the occurrence of [Ca2+](i) rise and increased 45Ca accumulation in cells exposed to 1 mM-CaCl2 after having been treated for 2 min with carbachol in Ca2+-free medium; (c) by typical differences in the quin2 signal kinetics observed in parallel samples of PC12 cells loaded with different concentrations of the dye.

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